P-Hydroxybenzoic Acid And Myistic Acid Modified Polymeric Micelles For Brain-targeting

Blood brain barrier (BBB) is a physical dynamic barrier between brain and blood, which serves as a protection barrier to effectively prevent certain foreign substances from entering the brain blood circulation. Due to poor permeability of the BBB, nearly100%of large molecules and98%of small molecules are forbidden access to central nervous system (CNS). Consequently, it hampers treatment and diagnosis of CNS diseases such as brain tumors, reduce Alzheimer’s diseases and Parkinson’s diseases.Glioblastoma multiforme (GBM) is the most common brain tumor, which accounts for approximately40%of the brain tumors. The commonly clinical treatment for the GBM involves surgical debulking of accessible tumor from the brain. However, the proximity to crotical regions for brain function makes complete removal of brain tumor very difficult, resulting in very high tumor recurrence. Therefore, chemotherapy seems essential for the post-surgical treatment of the GBM. To overcome the poor permeability of the BBB, researchers have been focusing on the development of novel nanocarriers to achieve brain targeted drug delivery.Receptor-mediated drug delivery systems represent very promising non-invasive strategy for active targeted drug delivery to the site of brain tumors. The specific receptors of the brain capillary endothelium include transferrin (Tf), insulin and monoclonal antibody receptors. However, there are some disadvantages associated with such large molecule ligands such as stability, immunogenicity and the complicated preparation process. Accordingly, novel brain-targeted delivery carriers based on small molecules such as p-hydroxybenzoic acid (p-HA) and myristic acid (MC) were developed in this study. The brain targeting ability of these two carriers were investigated in vitro and in vivo. Furthermore, anti-glioblastoma effect of these carriers was studied using docetaxel (DTX) as a model drug.The present studies includes three parts:(1) Preparation and characterization of brain-targeted polymeric micelles mediated by small moleculesp-HA and MC were conjugated with poly(ethylene glycol)-distearoylphosphatidylethanolamine (p-HA-PEG-DSPE and MC-PEG-DSPE) through acylation reaction, respectively. HPLC and’ H-NMR were used to determine the purity and structure of these two polymers. Polymeric micelles composed of methoxy-PEG-DSPE (mPEG-DSPE) or the combination of mPEG-DSPE, p-HA-PEG-DSPE and MC-PEG-DSPE were prepared via a film-hydration method. Transmission electron microscopy (TEM) and dynamic light scattering analysis showed that these micelles were uniform with a mean particle size around18nm±3nm. Encapsulation efficiencies of these micelles were83.5%±1.2%and drug loadings were7.7%±1.3%. Stability study indicated that these micelles were not stable in HEPES-buffered saline (pH7.4) stored at4℃. Therefore,2%mannitol was chosen as a lyoprotectant to prepare lyophilization formulation of these micelles. In vitro drug release testing showed that the micelles could achieve sustained DTX release.(2) Evaluation of in vitro and in vivo targeting ability and anti-glioblastoma effect of brain-targeted polymeric micellesCoumarin-6was loaded into the micelles to form mPEG-micelles/C6、 p-HA-micelles/C6和MC-micelles/C6, respectively. The micelles could avoid the macrophages endocytosis in vitro. Fluorescent microscopy and flow cytometer were used to evaluated in vitro cellular uptake of these micelles on the brain capillary endothelial cells (BCECs). p-HA-and MC-modified micelles showed improved cellular uptake by the BCECs cells compared to the unmodified micelles. In vivo and ex vivo near-infrared fluorescence imaging experiments demonstrated that brain targeting ability of the p-HA-and MC-modified micelles were1-2times higher than that of the unmodified micelles.In vitro cytotoxicity assay against U87MG glioblastoma cells showed that the p-HA-and MC-modified micelles loaded with DTX increased inhibition of tumor cell growth compared to that of the unmodified micelles and Taxotere(?), which indicated that the anti-glioblastoma effect of DTX could be enhanced by the p-HA-and MC-modified polymeric micelles. Anti-glioblastoma study in a xenograft mouse model of human glioblastoma multiforme (GBM) showed that both p-HA-and MC-conjugated micelles could significantly elongate the lifetime of U87grafted mice compared to saline, Taxotere(?) and mPEG-micelles groups (P<0.05).In the present study, novel brain-targeted PEG-DSPE micelles based on small molecule ligands (p-HA and MC) were developed. In vitro and in vivo targeting effect studies showed that both p-HA and MC could mediate the active brain-targeted delivery of the mPEG-DSPE micelles. Furthermore, p-HA and MC-modified micelles can improve the anti-gliobalstoma effect of DTX. These results demonstrated that the p-HA and MC modified PEG-DSPE micelles are promising brain-targeted drug delivery nanocarriers for hydrophobic anti-tumor therapeutics against glioblastoma.